Determining cells and allocating them to a cell type is very important in cytology. Cellular blood components, i.e. erythrocytes, thrombocytes and white blood cells, are determined and quantified in, for example, a hematological examination. Determination of the number of leucocytes (white blood count, WBC) should be able to differentiate the essential populations of the white blood cells for a comprehensive diagnosis. The white cells are differentiated according to granular cells (neutrophils, eosinophils, basophils) and non-granular cells (lymphocytes, monocytes). In addition to the granularity the cells also differ in respect of the fragmentation of the cell nucleus (without fragmentation: mononuclear cells, i.e. lymphocytes and monocytes; polymorphonuclear cells: eosinophils, basophils and neutrophils) and the cell size. A dye is used for the differentiation of the granular cells, in particular eosinophilic and basophilic granulocytes. The cell populations are conventionally evaluated by fully automatic hematology analyzers or by microscopy. Fully automatic analyzers have to analyze the populations according to fixed algorithms (with the aid of, for example, impedance, scattered light and absorption measurements). However, this often leads to error messages being displayed in the case of pathological samples, for example. In the next step microscopy conventionally takes place as a validation method for cells incorrectly determined by the hematology analyzer. This step is laborious and cost-intensive since it also requires manual evaluation in addition to sample preparation, microscopy and further work carried out manually.
It can also be important to determine the cell volume in addition to the morphology of the cell. Due to scattering by granules and polymorphic nuclei this is conventionally undertaken only in non-granular erythrocytes and platelets. Therefore, as a rule, this is not carried out for white blood cells.
In order, for example, to carry out quantitative blood cell diagnostics, a hematology analyzer can be dispensed with and instead each single cell can be examined using microscopy. This allows the blood count to be determined independently of fixed evaluation algorithms and flags. The drawback of this approach, however, is the lower sample throughput than with a hematology analyzer and the continued effort of immobilizing and dyeing the cells on microscope slides. These dyes also have only limited reproducibility, moreover, and exhibit a high dependency on humidity, dye life, temperature and more. A drawback of pure microscopy is quantifying the cell volumes compared to flow cytometers used in hematology analyzers.
An imaging flow cytometer is known from U.S. Pat. No. 8,406,498 whereby the differentiation of white blood cells can be determined directly. A dye is used in this case as well, however, to differentiate eosinophilic and basophilic granulocytes. The drawbacks mentioned above result here as well therefore.